PARticular MARks: Histone ADP-ribosylation and the DNA damage response.

Cellular and molecular responses to DNA damage are highly orchestrated and dynamic, acting to preserve the maintenance and integrity of the genome. Histone proteins bind DNA and organize the genome into chromatin. Post-translational modifications of histones have been shown to play an essential role in orchestrating the chromatin response to DNA damage by regulating the DNA damage response pathway. Among the histone modifications that contribute to this intricate network, histone ADP-ribosylation (ADPr) is emerging as a pivotal component of chromatin-based DNA damage response (DDR) pathways. In this review, we survey how histone ADPr is regulated to promote the DDR and how it impacts chromatin and other histone marks. Recent advancements have revealed histone ADPr effects on chromatin structure and the regulation of DNA repair factor recruitment to DNA lesions. Additionally, we highlight advancements in technology that have enabled the identification and functional validation of histone ADPr in cells and in response to DNA damage. Given the involvement of DNA damage and epigenetic regulation in human diseases including cancer, these findings have clinical implications for histone ADPr, which are also discussed. Overall, this review covers the involvement of histone ADPr in the DDR and highlights potential future investigations aimed at identifying mechanisms governed by histone ADPr that participate in the DDR, human diseases, and their treatments.

DNA methylation changes during a sprint interval exercise performed under normobaric hypoxia or with blood flow restriction: A pilot study in men.

This crossover study evaluated DNA methylation changes in human salivary samples following single sprint interval training sessions performed in hypoxia, with blood flow restriction (BFR), or with gravity-induced BFR. Global DNA methylation levels were evaluated with an enzyme-linked immunosorbent assay. Methylation-sensitive restriction enzymes were used to determine the percentage methylation in a part of the promoter of the gene-inducible nitric oxide synthase (p-iNOS), as well as an enhancer (e-iNOS). Global methylation increased after exercise (p < 0.001; dz = 0.50). A tendency was observed for exercise × condition interaction (p = 0.070). Post hoc analyses revealed a significant increase in global methylation between pre- (7.2 ± 2.6%) and postexercise (10.7 ± 2.1%) with BFR (p = 0.025; dz = 0.69). Methylation of p-iNOS was unchanged (p > 0.05). Conversely, the methylation of e-iNOS increased from 0.6 ± 0.4% to 0.9 ± 0.8% after exercise (p = 0.025; dz = 0.41), independently of the condition (p > 0.05). Global methylation correlated with muscle oxygenation during exercise (r = 0.37, p = 0.042), while e-iNOS methylation showed an opposite association (r = -0.60, p = 0.025). Furthermore, p-iNOS methylation was linked to heart rate (r = 0.49, p = 0.028). Hence, a single sprint interval training increases global methylation in saliva, and adding BFR tends to increase it further. Lower muscle oxygenation is associated with augmented e-iNOS methylation. Finally, increased cardiovascular strain results in increased p-iNOS methylation.

Neuronal identity control at the resolution of a single transcription factor isoform.

The brain exhibits remarkable neuronal diversity which is critical for its functional integrity. From the sheer number of cell types emerging from extensive transcriptional, morphological, and connectome datasets, the question arises of how the brain is capable of generating so many unique identities. 'Terminal selectors' are transcription factors hypothesized to determine the final identity characteristics in post-mitotic cells. Which transcription factors function as terminal selectors and the level of control they exert over different terminal characteristics are not well defined. Here, we establish a novel role for the transcription factor broad as a terminal selector in Drosophila melanogaster. We capitalize on existing large sequencing and connectomics datasets and employ a comprehensive characterization of terminal characteristics including Perturb-seq and whole-cell electrophysiology. We find a single isoform broad-z4 serves as the switch between the identity of two visual projection neurons LPLC1 and LPLC2. Broad-z4 is natively expressed in LPLC1, and is capable of transforming the transcriptome, morphology, and functional connectivity of LPLC2 cells into LPLC1 cells when perturbed. Our comprehensive work establishes a single isoform as the smallest unit underlying an identity switch, which may serve as a conserved strategy replicated across developmental programs.

Morphology and synapse topography optimize linear encoding of synapse numbers in Drosophila looming responsive descending neurons.

Synapses are often precisely organized on dendritic arbors, yet the role of synaptic topography in dendritic integration remains poorly understood. Utilizing electron microscopy (EM) connectomics we investigate synaptic topography in Drosophila melanogaster looming circuits, focusing on retinotopically tuned visual projection neurons (VPNs) that synapse onto descending neurons (DNs). Synapses of a given VPN type project to non-overlapping regions on DN dendrites. Within these spatially constrained clusters, synapses are not retinotopically organized, but instead adopt near random distributions. To investigate how this organization strategy impacts DN integration, we developed multicompartment models of DNs fitted to experimental data and using precise EM morphologies and synapse locations. We find that DN dendrite morphologies normalize EPSP amplitudes of individual synaptic inputs and that near random distributions of synapses ensure linear encoding of synapse numbers from individual VPNs. These findings illuminate how synaptic topography influences dendritic integration and suggest that linear encoding of synapse numbers may be a default strategy established through connectivity and passive neuron properties, upon which active properties and plasticity can then tune as needed.

Impact of systemic hypoxia and blood flow restriction on mechanical, cardiorespiratory, and neuromuscular responses to a multiple-set repeated sprint exercise.

Introduction: Repeated sprint cycling exercises (RSE) performed under systemic normobaric hypoxia (HYP) or with blood flow restriction (BFR) are of growing interest. To the best of our knowledge, there is no stringent consensus on the cardiorespiratory and neuromuscular responses between systemic HYP and BFR during RSE. Thus, this study assessed cardiorespiratory and neuromuscular responses to multiple sets of RSE under HYP or with BFR. Methods: According to a crossover design, fifteen men completed RSE (three sets of five 10-s sprints with 20 s of recovery) in normoxia (NOR), HYP, and with bilaterally-cuffed BFR at 45% of resting arterial occlusive pressure during sets in NOR. Power output, cardiorespiratory and neuromuscular responses were assessed. Results: Average peak and mean powers were lower in BFR (dz = 0.87 and dz = 1.23, respectively) and HYP (dz = 0.65 and dz = 1.21, respectively) compared to NOR (p < 0.001). The percentage decrement of power output was greater in BFR (dz = 0.94) and HYP (dz = 0.64) compared to NOR (p < 0.001), as well as in BFR compared to NOR (p = 0.037, dz = 0.30). The percentage decrease of maximal voluntary contraction of the knee extensors after the session was greater in BFR compared to NOR and HYP (p = 0.011, dz = 0.78 and p = 0.027, dz = 0.75, respectively). Accumulated ventilation during exercise was higher in HYP and lower in BFR (p = 0.002, dz = 0.51, and p < 0.001, dz = 0.71, respectively). Peak oxygen consumption was reduced in HYP (p < 0.001, dz = 1.47). Heart rate was lower in BFR during exercise and recovery (p < 0.001, dz = 0.82 and p = 0.012, dz = 0.43, respectively). Finally, aerobic contribution was reduced in HYP compared to NOR (p = 0.002, dz = 0.46) and BFR (p = 0.005, dz = 0.33). Discussion: Thus, this study indicates that power output during RSE is impaired in HYP and BFR and that BFR amplifies neuromuscular fatigue. In contrast, HYP did not impair neuromuscular function but enhanced the ventilatory response along with reduced oxygen consumption.

Design, synthesis and validation of a new Crimped Head-Piece for DNA-Encoded libraries generation.

Codification of DNA Encoded Libraries (DELs) is critical for successful ligand identification of molecules that bind a protein of interest (POI). There are different encoding strategies that permit, for instance, the customization of a DEL for testing single or dual pharmacophores (single strand DNA) or for producing and screening large diversity libraries of small molecules (double strand DNA). Both approaches challenges, either from the synthetic and encoding point of view, or from the selection methodology to be utilized for the screening. The Head-Piece contains the DNA sequence that is attached to a chemical compound, allowing the encoding of each molecule with a unique DNA tag. Designing the Head-Piece for a DNA-encoded library involves careful consideration of several key aspects including DNA barcode identity, sequence length and attachment chemistry. Here we describe a double stranded DNA versatile Head-Piece that can be used for the generation of single or dual pharmacophore libraries, but also shows other advanced DEL functionalities, stability and enlarged encoding capacity.

APOBEC3B regulates R-loops and promotes transcription-associated mutagenesis in cancer.

The single-stranded DNA cytosine-to-uracil deaminase APOBEC3B is an antiviral protein implicated in cancer. However, its substrates in cells are not fully delineated. Here APOBEC3B proteomics reveal interactions with a surprising number of R-loop factors. Biochemical experiments show APOBEC3B binding to R-loops in cells and in vitro. Genetic experiments demonstrate R-loop increases in cells lacking APOBEC3B and decreases in cells overexpressing APOBEC3B. Genome-wide analyses show major changes in the overall landscape of physiological and stimulus-induced R-loops with thousands of differentially altered regions, as well as binding of APOBEC3B to many of these sites. APOBEC3 mutagenesis impacts genes overexpressed in tumors and splice factor mutant tumors preferentially, and APOBEC3-attributed kataegis are enriched in RTCW motifs consistent with APOBEC3B deamination. Taken together with the fact that APOBEC3B binds single-stranded DNA and RNA and preferentially deaminates DNA, these results support a mechanism in which APOBEC3B regulates R-loops and contributes to R-loop mutagenesis in cancer.

OTUD5 limits replication fork instability by organizing chromatin remodelers.

Proper regulation of replication fork progression is important for genomic maintenance. Subverting the transcription-induced conflicts is crucial in preserving the integrity of replication forks. Various chromatin remodelers, such as histone chaperone and histone deacetylases are known to modulate replication stress, but how these factors are organized or collaborate are not well understood. Here we found a new role of the OTUD5 deubiquitinase in limiting replication stress. We found that OTUD5 is recruited to replication forks, and its depletion causes replication fork stress. Through its C-terminal disordered tail, OTUD5 assembles a complex containing FACT, HDAC1 and HDAC2 at replication forks. A cell line engineered to specifically uncouple FACT interaction with OTUD5 exhibits increases in FACT loading onto chromatin, R-loop formation, and replication fork stress. OTUD5 mediates these processes by recruiting and stabilizing HDAC1 and HDAC2, which decreases H4K16 acetylation and FACT recruitment. Finally, proteomic analysis revealed that the cells with deficient OTUD5-FACT interaction activates the Fanconi Anemia pathway for survival. Altogether, this study identified a new interaction network among OTUD5-FACT-HDAC1/2 that limits transcription-induced replication stress.

Active recovery vs hot- or cold-water immersion for repeated sprint ability after a strenuous exercise training session in elite skaters.

This study compared the acute effects of three recovery methods: active recovery (AR), hot- and cold-water immersion (HWI and CWI, respectively), used between two training sessions in elite athletes. Twelve national-team skaters (7 males, 5 females) completed three trials according to a randomized cross-over study. Fifteen minutes after an exhaustive ice-skating training session, participants underwent 20 min of HWI (41.1 ± 0.5°C), 15 min of CWI (12.1 ± 0.7°C) or 15 min of active recovery (AR). After 1 h 30 min of the first exercise, they performed a repeated-sprint cycling session. Average power output was slightly but significantly higher for AR (767 ± 179 W) and HWI (766 ± 170 W) compared to CWI (738 ± 156 W) (p = 0.026, d = 0.18). No statistical difference was observed between the conditions for both lactatemia and rating of perceived exertion. Furthermore, no significant effect of recovery was observed on the fatigue index calculated from the repeated sprint cycling exercises (p > 0.05). Finally, a positive correlation was found between the average muscle temperature measured during the recoveries and the maximal power output obtained during cycling exercises. In conclusion, the use of CWI in between high-intensity training sessions could slightly impair the performance outcomes compared to AR and HWI. However, studies with larger samples are needed to confirm these results, especially in less trained athletes.

Effects of short-term repeated sprint training in hypoxia or with blood flow restriction on response to exercise.

This study compared the effects of a brief repeated sprint training (RST) intervention performed with bilateral blood flow restriction (BFR) conditions in normoxia or conducted at high levels of hypoxia on response to exercise. Thirty-nine endurance-trained athletes completed six repeated sprints cycling sessions spread over 2 weeks consisting of four sets of five sprints (10-s maximal sprints with 20-s active recovery). Athletes were assigned to one of the four groups and subjected to a bilateral partial blood flow restriction (45% of arterial occlusion pressure) of the lower limbs during exercise (BFRG), during the recovery (BFRrG), exercised in a hypoxic room simulating hypoxia at FiO2 ≈ 13% (HG) or were not subjected to additional stress (CG). Peak aerobic power during an incremental test, exercise duration, maximal accumulated oxygen deficit and accumulated oxygen uptake (VO2) during a supramaximal constant-intensity test were improved thanks to RST (p < 0.05). No significant differences were observed between the groups (p > 0.05). No further effect was found on other variables including time-trial performance and parameters of the force-velocity relationship (p > 0.05). Thus, peak aerobic power, exercise duration, maximal accumulated oxygen deficit, and VO2 were improved during a supramaximal constant-intensity exercise after six RST sessions. However, combined hypoxic stress or partial BFR did not further increase peak aerobic power.

Muscle Deoxygenation Rates and Reoxygenation Modeling During a Sprint Interval Training Exercise Performed Under Different Hypoxic Conditions.

This study compared the kinetics of muscle deoxygenation and reoxygenation during a sprint interval protocol performed under four modalities: blood flow restriction at 60% of the resting femoral artery occlusive pressure (BFR), gravity-induced BFR (G-BFR), simulated hypoxia (FiO2≈13%, HYP) and normoxia (NOR). Thirteen healthy men performed each session composed of five all-out 30-s efforts interspaced with 4 min of passive recovery. Total work during the exercises was 17 ± 3.4, 15.8 ± 2.9, 16.7 ± 3.4, and 18.0 ± 3.0 kJ for BFR, G-BFR, HYP and NOR, respectively. Muscle oxygenation was continuously measured with near-infrared spectroscopy. Tissue saturation index (TSI) was modelled with a linear function at the beginning of the sprint and reoxygenation during recovery with an exponential function. Results showed that both models were adjusted to the TSI (R2 = 0.98 and 0.95, respectively). Greater deoxygenation rates were observed in NOR compared to BFR (p = 0.028). No difference was found between the conditions for the deoxygenation rates relative to sprint total work (p > 0.05). Concerning reoxygenation, the amplitude of the exponential was not different among conditions (p > 0.05). The time delay of reoxygenation was longer in BFR compared to the other conditions (p < 0.05). A longer time constant was found for G-BFR compared to the other conditions (p < 0.05), and mean response time was longer for BFR and G-BFR. Finally, sprint performance was correlated with faster reoxygenation. Hence, deoxygenation rates were not different between the conditions when expressed relatively to total sprint work. Furthermore, BFR conditions impair reoxygenation: BFR delays and G-BFR slows down reoxygenation.

NAMPT Inhibition Induces Neuroblastoma Cell Death and Blocks Tumor Growth.

High-risk neuroblastoma (NB) portends very poor prognoses in children. Targeting tumor metabolism has emerged as a novel therapeutic strategy. High levels of nicotinamide-adenine-dinucleotide (NAD+) are required for rapid cell proliferation. Nicotinamide phosphoribosyl transferase (NAMPT) is the rate-limiting enzyme for NAD+ salvage and is overexpressed in several cancers. Here, we determine the potential of NAMPT as a therapeutic target for NB treatment. NAMPT inhibition cytotoxicity was determined by trypan blue exclusion and LDH assays. Neuroblastoma stem cell self-renewal was evaluated by neurosphere assay. Protein expression was evaluated via Western blot. The effect of targeting NAMPT in vivo was determined using an NB1691-xenografted mouse model. Robust NAMPT expression was demonstrated in multiple N-MYC amplified, high-risk neuroblastoma cell lines. NAMPT inhibition with STF-118804 (STF) decreased ATP, induced apoptosis, and reduced NB stem cell neurosphere formation. STF treatment down-regulated N-MYC levels and abrogated AKT activation. AKT and glycolytic pathway inhibitors in combination with NAMPT inhibition induced robust, greater-than-additive neuroblastoma cell death. Lastly, STF treatment blocked neuroblastoma tumor growth in mouse xenograft models. NAMPT is a valid therapeutic target as inhibition promoted neuroblastoma cell death in vitro and prevented tumor growth in vivo. Further investigation is warranted to establish this therapy's role as an adjunctive modality.

Joining the PARty: PARP Regulation of KDM5A during DNA Repair (and Transcription?).

The lysine demethylase KDM5A collaborates with PARP1 and the histone variant macroH2A1.2 to modulate chromatin to promote DNA repair. Indeed, KDM5A engages poly(ADP-ribose) (PAR) chains at damage sites through a previously uncharacterized coiled-coil domain, a novel binding mode for PAR interactions. While KDM5A is a well-known transcriptional regulator, its function in DNA repair is only now emerging. Here we review the molecular mechanisms that regulate this PARP1-macroH2A1.2-KDM5A axis in DNA damage and consider the potential involvement of this pathway in transcription regulation and cancer. Using KDM5A as an example, we discuss how multifunctional chromatin proteins transition between several DNA-based processes, which must be coordinated to protect the integrity of the genome and epigenome. The dysregulation of chromatin and loss of genome integrity that is prevalent in human diseases including cancer may be related and could provide opportunities to target multitasking proteins with these pathways as therapeutic strategies.

Combined noncanonical NF-κB agonism and targeted BET bromodomain inhibition reverse HIV latency ex vivo.

Latency reversal strategies for HIV cure using inhibitor of apoptosis protein (IAP) antagonists (IAPi) induce unprecedented levels of latent reservoir expression without immunotoxicity during suppressive antiretroviral therapy (ART). However, full targeting of the reservoir may require combinatorial approaches. A Jurkat latency model screen for IAPi combination partners demonstrated synergistic latency reversal with bromodomain (BD) and extraterminal domain protein inhibitors (BETi). Mechanistic investigations using CRISPR-CAS9 and single-cell RNA-Seq informed comprehensive ex vivo evaluations of IAPi plus pan-BET, bD-selective BET, or selective BET isoform targeting in CD4+ T cells from ART-suppressed donors. IAPi+BETi treatment resulted in striking induction of cell-associated HIV gag RNA, but lesser induction of fully elongated and tat-rev RNA compared with T cell activation-positive controls. IAPi+BETi resulted in HIV protein induction in bulk cultures of CD4+ T cells using an ultrasensitive p24 assay, but did not result in enhanced viral outgrowth frequency using a standard quantitative viral outgrowth assay. This study defines HIV transcriptional elongation and splicing as important barriers to latent HIV protein expression following latency reversal, delineates the roles of BET proteins and their BDs in HIV latency, and provides a rationale for exploration of IAPi+BETi in animal models of HIV latency.

Cardiac Arrest and Hypoxic-Ischemic Encephalopathy Following an Emergency Caesarean Section in a COVID-19 Patient.

While young, healthy individuals without underlying medical conditions have generally not suffered catastrophic health consequences from the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), gravid patients appear to be at much higher risk of complications from this virus. A 29-year-old G3P2 patient at 30 weeks and three days presented with worsening dyspnea and chest pain after testing positive for coronavirus disease 2019 (COVID-19) infection two days prior. Notably, she had not received COVID-19 vaccination. A non-reassuring fetal tracing and fetal bradycardia were discovered on routine prenatal monitoring during admission, and an urgent caesarean section was performed. She subsequently required supplemental oxygen due to respiratory distress and remained hospitalized. She clinically deteriorated from a respiratory standpoint. Several days later, she experienced cardiac arrest with a return of spontaneous circulation (ROSC) in nine minutes. While the baby was discharged home and is doing well, the patient, unfortunately, expired from hypoxic encephalopathy secondary to COVID-19 pneumonia and complications of cardiorespiratory arrest. This case highlights the severe sequelae of COVID-19 infection in a postpartum patient, including ventilator-dependent respiratory failure, sudden cardiac death, hypoxic encephalopathy, and coma.

Transmission of COVID-19 From an Asymptomatic Mother to Fetus Leading to Death of Neonate From Pneumonia.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) responsible for the coronavirus disease 2019 (COVID-19) pandemic has rarely impacted neonates. When infection does occur, it is typically asymptomatic. We describe a case of a neonate born to a 25-year-old mother who was COVID-19 positive but asymptomatic. An emergent cesarean section was performed during week 30 of gestation due to category three fetal heart tracings. The neonate, unfortunately, died on the day of life 12 from respiratory distress secondary to severe COVID-19 pneumonia. This is an important case that illustrates the deleterious impact COVID-19 infection can have on neonates. It is a unique case of the compassionate use of remdesivir for a neonate. The patient's respiratory decline soon after birth, lends support that the virus responsible for COVID-19 can be transmitted vertically.

Unveiling the Chemical Diversity of the Deep-Sea Sponge Characella pachastrelloides.

Sponges are at the forefront of marine natural product research. In the deep sea, extreme conditions have driven secondary metabolite pathway evolution such that we might expect deep-sea sponges to yield a broad range of unique natural products. Here, we investigate the chemodiversity of a deep-sea tetractinellid sponge, Characella pachastrelloides, collected from ~800 m depth in Irish waters. First, we analyzed the MS/MS data obtained from fractions of this sponge on the GNPS public online platform to guide our exploration of its chemodiversity. Novel glycolipopeptides named characellides were previously isolated from the sponge and herein cyanocobalamin, a manufactured form of vitamin B12, not previously found in nature, was isolated in a large amount. We also identified several poecillastrins from the molecular network, a class of polyketide known to exhibit cytotoxicity. Light sensitivity prevented the isolation and characterization of these polyketides, but their presence was confirmed by characteristic NMR and MS signals. Finally, we isolated the new betaine 6-methylhercynine, which contains a unique methylation at C-2 of the imidazole ring. This compound showed potent cytotoxicity towards against HeLa (cervical cancer) cells.

Making Connections: Integrative Signaling Mechanisms Coordinate DNA Break Repair in Chromatin.

DNA double-strand breaks (DSBs) are hazardous to genome integrity and can promote mutations and disease if not handled correctly. Cells respond to these dangers by engaging DNA damage response (DDR) pathways that are able to identify DNA breaks within chromatin leading ultimately to their repair. The recognition and repair of DSBs by the DDR is largely dependent on the ability of DNA damage sensing factors to bind to and interact with nucleic acids, nucleosomes and their modified forms to target these activities to the break site. These contacts orientate and localize factors to lesions within chromatin, allowing signaling and faithful repair of the break to occur. Coordinating these events requires the integration of several signaling and binding events. Studies are revealing an enormously complex array of interactions that contribute to DNA lesion recognition and repair including binding events on DNA, as well as RNA, RNA:DNA hybrids, nucleosomes, histone and non-histone protein post-translational modifications and protein-protein interactions. Here we examine several DDR pathways that highlight and provide prime examples of these emerging concepts. A combination of approaches including genetic, cellular, and structural biology have begun to reveal new insights into the molecular interactions that govern the DDR within chromatin. While many questions remain, a clearer picture has started to emerge for how DNA-templated processes including transcription, replication and DSB repair are coordinated. Multivalent interactions with several biomolecules serve as key signals to recruit and orientate proteins at DNA lesions, which is essential to integrate signaling events and coordinate the DDR within the milieu of the nucleus where competing genome functions take place. Genome architecture, chromatin structure and phase separation have emerged as additional vital regulatory mechanisms that also influence genome integrity pathways including DSB repair. Collectively, recent advancements in the field have not only provided a deeper understanding of these fundamental processes that maintain genome integrity and cellular homeostasis but have also started to identify new strategies to target deficiencies in these pathways that are prevalent in human diseases including cancer.